1. Field of the Invention
The present invention relates generally to the field of molecular biology. More specifically, the invention relates to plant fatty acid amide hydrolase genes and methods of use thereof.
2. Description of the Related Art
N-Acylethanolamines (NAEs) are endogenous constituents of plant and animal tissues, and in vertebrates their hydrolysis terminates their participation as lipid mediators in the endocannabinoid signaling system. The membrane-bound enzyme responsible for NAE hydrolysis in mammals has been identified at the molecular level (designated fatty acid amide hydrolase, FAAH), and although an analogous enzyme activity was identified in microsomes of cotton seedlings, no molecular information has been available for this enzyme in plants.
NAEs are produced from the hydrolysis of N-acylphosphatidylethanolamines (NAPEs), a minor membrane lipid constituent of cellular membranes, by phospholipase D in animal systems (Schmid et al., 1996). One example of an NAE, anandamide (NAE 20:4), has varied physiological roles as an endogenous ligand for cannabinoid receptors and functions in modulation of neurotransmission in the central nervous system (Wilson and Nicoll, 2002). Anandamide also activates vanilloid receptors and functions as an endogenous analgesic (Pertwee, 2001) and appears to be involved in neuroprotection (Hansen et al., 2000; Van der Stelt et al., 2001). While a principal role for NAE20:4 as an endogenous ligand for cannabinoid receptors has emerged as a paradigm for endocannabinoid signaling (Desarnaud et al., 1995; Wilson and Nicoll, 2002), other types of NAEs as well as other fatty acid derivatives likely interact with this pathway and perhaps others directly or indirectly to modulate a variety of physiological functions in vertebrates (Lambert and Di Marzo, 1999; Lambert et al., 2002; Schmid and Berdyshev, 2002; Schmid et al., 2002).
NAEs have been implicated in immunomodulation (Buckley et al., 2000), synchronization of embryo development (Paria and Dey, 2000), and induction of apoptosis (Sarker et al., 2000). These endogenous bioactive molecules lose their signaling activity upon hydrolysis by fatty acid amide hydrolase (FAAH). Advances in the understanding of FAAH function in mammals at the structural level (Bracey et al., 2002), mechanistic level, and the physiological level (knockouts), have been made possible only through the cloning, expression and manipulation of the cDNA/gene encoding FAAH (Giang and Cravatt, 1997). Such studies have been lacking in plants due to the failure to isolate identify FAAH genes.
Research in the last decade has, however, indicated that NAE metabolism occurs in plants by pathways analogous to those in vertebrates and invertebrates (Chapman, 2000, Shrestha et al., 2002), pointing to the possibility that these lipids may be an evolutionarily conserved mechanism for the regulation of physiology in multicellular organisms. In plants, NAEs are present in substantial amounts in desiccated seeds (˜1 μg g−1 fresh wt) and their levels decline after a few hours of imbibition (Chapman et al., 1999). Individual plant NAEs have been identified in plants as predominantly 16C and 18C species with N-palmitoylethanolamine (NAE 16:0) and N-linoleoylethanolamine (NAE 18:2) generally being the most abundant. Like in animal cells, plant NAEs are derived from N-acylphosphatidylethanolamines (NAPEs) (Schmid et al., 1990; Chapman, 2000) by the action of a phospholipase D (PLD). The occurrence of NAEs in seeds and their rapid depletion during seed imbibition (Chapman, 2000) suggests that these lipids may have a role in the regulation of seed germination.
Recently, depletion of NAEs during seed imbibiton/germination was determined to occur via two metabolic pathways—one lipoxygenase—mediated, for the formation of NAE oxylipins from NAE 18:2, and one amidase—mediated for hydrolysis of saturated and unsaturated NAEs (Shrestha et al., 2002). Hydrolysis of NAEs was reconstituted and characterized in microsomes of cottonseeds, and appeared to be catalyzed by an enzyme similar to the FAAH of mammalian species (Shrestha et al., 2002).
While the foregoing studies have provided a further understanding of the metabolism of plant secondary metabolism, the prior art has failed to provide genes encoding plant fatty acid amide hydrolase. The identification of such genes would allow the creation of novel plants with improved phenotypes and methods for use thereof. There is, therefore, a great need in the art for the identification of plant fatty acid amide hydrolase genes.